EP1339873B1 - Diagnostic de maladies associees au gene c-mos humain - Google Patents

Diagnostic de maladies associees au gene c-mos humain Download PDF

Info

Publication number
EP1339873B1
EP1339873B1 EP20010982464 EP01982464A EP1339873B1 EP 1339873 B1 EP1339873 B1 EP 1339873B1 EP 20010982464 EP20010982464 EP 20010982464 EP 01982464 A EP01982464 A EP 01982464A EP 1339873 B1 EP1339873 B1 EP 1339873B1
Authority
EP
European Patent Office
Prior art keywords
dna
seq
humos
gene
fragments
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP20010982464
Other languages
German (de)
English (en)
Other versions
EP1339873A2 (fr
Inventor
Alexander Olek
Christian Piepenbrock
Kurt Berlin
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Epigenomics AG
Original Assignee
Epigenomics AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Epigenomics AG filed Critical Epigenomics AG
Publication of EP1339873A2 publication Critical patent/EP1339873A2/fr
Application granted granted Critical
Publication of EP1339873B1 publication Critical patent/EP1339873B1/fr
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to nucleic acids and to a method of diagnosing diseases related to the genetic and / or epigenetic parameters of the oncogene humos and in particular its methylation status.
  • Moloney murine sarcoma virus is one of the replication-inactive retroviruses that transforms fibroblasts into culture and induces sarcomas in vivo.
  • the virus was created by the recombination between the Moloney murine leukemia virus and a mouse cell-derived sequence.
  • the murine cell-derived segment of MSV also known as v-mos, is required for the induction and maintenance of viral transformation.
  • Homologous genes to v-mos are mouse-derived c-mos and human c-mos (humos), which was mapped to the human chromosome 8q11-12, thanks to the evolutionary conservation of viral oncogenes among vertebrate species (Prakash K , McBride OW, Swan DC, Devare SG, Tronick SR, Aaronson SA Molecular cloning and chromosomal mapping of a human locus related to the transforming Gene of Moloney murine sarcoma virus. Proc Natl Acad Sci USA 1982 Sep; 79 (17): 5210-4; Watson R, Oskarsson M, Vande Woude GF.
  • c-mos Human DNA sequence homologous to the transforming gene (mos) of Moloney murine sarcoma virus. Proc Natl Acad Sci USA. 1982 Jul; 79 (13): 4078-82.).
  • the c-mos proto-oncogene is expressed in a significant number of lung carcinomas and may play a role in their development (Athanasiou A, Gorgoulis VG, Zacharatos P, Mariatos G, Kotsinas A, Liloglou T, Karameris A, Foukas P, Manolis EN , Field JK, Kittas C.
  • c-mos immunoreactivity is an indicator of good prognosis in lung cancer., Histopathology., 2000 Jul; 37 (1): 45-54).
  • 5-methylcytosine is the most abundant covalently modified base in the DNA of eukaryotic cells. For example, it plays a role in the regulation of transcription, genetic imprinting and tumorigenesis. The identification of 5-methylcytosine as a component of genetic information is therefore of considerable interest. However, 5-methylcytosine positions can not be identified by sequencing because 5-methylcytosine has the same base-pairing behavior as cytosine. Moreover, in a PCR amplification, the epigenetic information carrying the 5-methylcytosines is completely lost.
  • a relatively new and now most commonly used method for studying DNA for 5-methylcytosine is based on the specific reaction of bisulfite with cytosine, which, after subsequent alkaline hydrolysis, is converted to uracil, which corresponds to thymidine in its base pairing behavior.
  • 5-methylcytosine is not modified under these conditions. This is how the original DNA is transformed that methylcytosine, which can not initially be distinguished from cytosine by its hybridization behavior, can now be detected by "normal" molecular biology techniques as the only remaining cytosine, for example by amplification and hybridization or sequencing. All of these techniques are based on base pairing which is now fully exploited.
  • the state of the art in terms of sensitivity is defined by a method involving the DNA to be tested in an agarose matrix, thereby preventing the diffusion and renaturation of the DNA (bisulfite reacts only on single-stranded DNA) and all precipitation and purification steps replaced by rapid dialysis (Olek A, Oswald J, Walter J. A modified and improved method for bisulphite-based cytosine methylation analysis, Nucleic Acids Res. 1996 Dec 15; 24 (24): 5064-6). With this method, individual cells can be examined, which illustrates the potential of the method. However, so far only single regions are investigated to about 3000 base pairs in length, a global investigation of cells on thousands of possible methylation analyzes is not possible.
  • the bisulfite technique is so far with a few exceptions (eg Zeschnigk M, Lich C, Buiting K, Doerfler W, Horsthemke B. A single-tube PCR test for the diagnosis of Angelman and Prader-Willi syndrome based on allelic methylation differences at the SNRPN locus. Eur J Hum Genet. 1997 Mar-Apr; 5 (2): 94-8) only used in research. However, short, specific pieces of a known gene are always amplified after bisulfite treatment and either completely sequenced (Olek A, Walter J.
  • Genomic sequencing indicates a correlation between DNA hypomethylation in the 5 'region of the pS2 gene and its expression in human breast cancer cell lines. Genes. 1995 May 19; 157 (1-2): 261-4; WO 97 46705, WO 95 15373 and WO 97/45560.
  • fluorescence-labeled probes have been used for the scanning of an immobilized DNA array.
  • fluorescent labels is the simple attachment of Cy3 and Cy5 dyes to the 5'-OH of the respective probe.
  • the detection of the fluorescence of the hybridized probes takes place for example via a confocal microscope.
  • the dyes Cy3 and Cy5 are commercially available, among many others.
  • Matrix-assisted laser desorption / ionization mass spectrometry is a very powerful development for the analysis of biomolecules (Karas M, Hillenkamp F. Laser desorption ionization of proteins with molecular masses 10,000 daltons. Anal Chem. 1988 Oct 15; 60 (20): 2299-301).
  • An analyte is embedded in a light-absorbing matrix.
  • a short laser pulse vaporizes the matrix and transports the analyte molecule un-fragmented into the gas phase. By collisions with matrix molecules, the ionization of the analyte is achieved.
  • An applied voltage accelerates the ions into a field-free flight tube. Due to their different masses ions are accelerated at different speeds. Smaller ions reach the detector earlier than larger ones.
  • MALDI-TOF spectrometry is excellently suited for the analysis of peptides and proteins. Analysis of nucleic acids is somewhat more difficult (Gut I G, Beck S. DNA and Matrix Assisted Laser Desorption Ionization Mass Spectrometry, Current Innovations and Future Trends., 1995, 1; 147-57). For nucleic acids, the sensitivity is about 100 times worse than for peptides and decreases disproportionately with increasing fragment size. For nucleic acids that have a multiply negatively charged backbone, the ionization process through the matrix is much less efficient. The choice of matrix plays an eminently important role in MALDI-TOF spectrometry.
  • Genomic DNA is obtained by standard methods from DNA from cell, tissue or other experimental samples. This standard methodology can be found in references such as Fritsch and Maniatis eds., Molecular Cloning: A Laboratory Manual, 1989.
  • the present disclosure describes oligonucleotides and / or PNA oligomers for the detection of cytosine methylations and provides a method which is particularly useful for the diagnosis of genetic and epigenetic parameters of the humos gene.
  • the invention is based on the finding that, in particular, cytosine methylation patterns are particularly suitable for the diagnosis of diseases associated with humus.
  • the invention is based on the finding that genetic and epigenetic parameters and in particular the cytosine methylation pattern of the humos gene are particularly suitable for the diagnosis of diseases associated with humus.
  • the present disclosure further describes an oligonucleotide or oligomer for detecting the cytosine methylation state in chemically pretreated DNA comprising at least one base sequence of at least 13 nucleotides in length attached to a chemically pretreated DNA of the gene humos according to any one of Seq. ID No.1 to Seq. ID No.4 hybridizes.
  • the oligomer probes are important and effective tools that enable the identification of genetic and epigenetic parameters of the humos gene.
  • the base sequence of the oligomers comprises at least one CpG dinucleotide.
  • the probes may also be in the form of a PNA (Peptide Nucleic Acid) which has particularly preferred mating properties.
  • oligonucleotides according to the invention in which the cytosine of the CpG dinucleotide is the 5th to 9th nucleotide from the 5 'end of the 13-mer, in the case of PNA oligomers it is preferred that the cytosine of the CpG dinucleotide be the 4th 6. nucleotide from the 5 'end of the 9 mers is.
  • the oligomers are normally used in so-called sets, which for each of the CpG dinucleotides one of the sequences of Seq. ID No.1 to Seq. ID No.4 comprise at least one oligomer.
  • a set is preferred which comprises at least one oligomer for each of the CpG dinucleotides from one of Seq ID No.1 to Seq ID No.4.
  • the disclosure describes a set of at least two oligonucleotides which are used as so-called primer oligonucleotides for the amplification of DNA sequences of one of Seq. ID No.1 to Seq. ID No.4 or sections thereof.
  • At least one oligonucleotide is bound to a solid phase.
  • the present disclosure further describes a set of at least 10 oligomers (oligonucleotides and / or PNA oligomers) which serve to detect the cytosine methylation state in chemically pretreated genomic DNA (SEQ ID No.1 to SEQ ID NO: 4). These probes allow the diagnosis of genetic and epigenetic parameters of the humos gene.
  • the set of oligomers can also be used to detect single nucleotide polymorphisms (SNPs) in the chemically pretreated DNA of the gene humos according to one of Seq. ID No.1 to Seq. ID No.4 can be used.
  • a described arrangement of different oligonucleotides and / or PNA oligomers is bound to a solid phase.
  • This array of different oligonucleotide and / or PNA oligomer sequences may be characterized by being arranged on the solid phase in the form of a rectangular or hexagonal lattice.
  • the solid phase surface consists of silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver or gold.
  • nitrocellulose and plastics such as nylon, which may be in the form of spheres or as resin matrices.
  • Another object of the invention relates to the use of a DNA chip for analysis in connection with h ⁇ mos associated diseases, according to claim 16.
  • DNA chips are known for example from US 5,837,832.
  • a kit is further described which comprises, for example, a bisulfite-containing reagent, a set of primer oligonucleotides comprising at least two oligonucleotides whose sequences each have at least one 18-base-pair portion of the base sequences listed in the appendix (SEQ ID Nos. 1 to SEQ ID NO .4) or are complementary to them, oligonucleotides and / or PNA oligomers and a guide to the implementation and evaluation of the described method may exist.
  • a kit may contain only parts of the aforementioned ingredients.
  • the invention further provides a method of detecting genetic and / or epigenetic parameters of the humos gene by analysis of cytosine methylations and single nucleotide polymorphisms according to claim 1, comprising the steps of:
  • a genomic DNA sample is chemically treated in such a way that unmethylated cytosine bases are converted into uracil, thymine or another base which is dissimilar to cytosine by the hybridization behavior at the 5'-position. This will be understood below as chemical pretreatment.
  • the genomic DNA to be analyzed is preferably obtained from the usual sources of DNA, such as cells or cell components, for example cell lines, biopsins, blood, sputum, stool, urine, cerebrospinal fluid, paraffin-embedded tissue, for example tissue of the eyes, Intestine, kidney, brain, heart, prostate, lung, breast or liver, histological slides or combinations thereof.
  • sources of DNA such as cells or cell components, for example cell lines, biopsins, blood, sputum, stool, urine, cerebrospinal fluid, paraffin-embedded tissue, for example tissue of the eyes, Intestine, kidney, brain, heart, prostate, lung, breast or liver, histological slides or combinations thereof.
  • genomic DNA fragments are amplified using sets of primer oligonucleotides of the invention and a preferably heat stable polymerase. For statistical and practical considerations, preferably more than ten different fragments are amplified which are 100-2,000 base pairs long.
  • the amplification of several DNA sections can be done simultaneously in one and the same reaction vessel. Usually, the amplification is carried out by means of the polymerase chain reaction (PCR).
  • the set of primer oligonucleotides comprises at least two oligonucleotides whose sequences are each reverse-complementary or identical to an at least 18 base-pair section of the base sequences listed in the Appendix (SEQ ID No.1 to SEQ ID No.4).
  • the primer oligonucleotides are preferably characterized by containing no CpG dinucleotide.
  • At least one primer oligonucleotide prefferably bound to a solid phase during the amplification.
  • the different oligonucleotide and / or PNA oligomer sequences can be arranged on a plane solid phase in the form of a rectangular or hexagonal lattice, the solid phase surface preferably consisting of silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver or gold, although other materials such as nitrocellulose or plastics can be used.
  • the fragments obtained by means of the amplification can carry a directly or indirectly detectable label.
  • the detection can be performed and visualized by matrix assisted laser desorption / ionization mass spectrometry (MALDI) or by electrospray mass spectrometry (ESI).
  • MALDI matrix assisted laser desorption / ionization mass spectrometry
  • ESI electrospray mass spectrometry
  • the amplificates obtained in the second method step are then hybridized to a set of oligonucleotides and / or PNA probes or to an array.
  • the hybridization is carried out in the manner indicated below.
  • the set used in the hybridization preferably consists of at least 10 oligonucleotide or PNA-oligomer probes.
  • the amplificates serve as probes which hybridize to previously bound to a solid phase oligonucleotides.
  • the unhybridized fragments are then removed.
  • Said oligonucleotides comprise at least one base sequence of 13 nucleotides in length which is reverse complementary or identical to a segment the base sequences listed in the appendix containing at least one CpG dinucleotide.
  • the cytosine of the CpG dinucleotide is considered to be the 5th to the 9th nucleotide from the 5 'end of the 13-mer.
  • an oligonucleotide is present for each CpG dinucleotide.
  • Said PNA oligomers comprise at least one 9-nucleotide base sequence which is reverse-complementary or identical to a portion of the base sequences listed in the Appendix containing at least one CpG dinucleotide.
  • the cytosine of the CpG dinucleotide is the 4th to 6th nucleotides seen from the 5 'end of the 9-mer.
  • an oligonucleotide is present for each CpG dinucleotide an oligonucleotide is present.
  • the unhybridized amplificates are removed.
  • the hybridized amplificates are detected. It is preferred that labels attached to the amplificates are identifiable at each position of the solid phase at which an oligonucleotide sequence is located.
  • the labels of the amplificates are fluorescent labels, radionuclides or detachable molecular fragments with typical mass, which can be detected in a mass spectrometer. Detection of the amplificates, fragments of the amplificates or probes complementary to the amplificates in the mass spectrometer is preferred, whereby the detection can be carried out and visualized by means of matrix assisted laser desorption / ionization mass spectrometry (MALDI) or by electrospray mass spectrometry (ESI).
  • MALDI matrix assisted laser desorption / ionization mass spectrometry
  • ESI electrospray mass spectrometry
  • the generated fragments may have a single positive or negative net charge.
  • the aforementioned method is used to determine genetic and / or epigenetic parameters of the humos gene.
  • the oligomers or arrays thereof are used to diagnose a humic-associated disease by analysis of methylation patterns of the humos gene.
  • Preferred according to the invention is the use of the method for the diagnosis of significant genetic and / or epigenetic parameters within the humos gene.
  • the nucleic acids of Seq. ID No.1 to Seq. ID No.4 can be used to diagnose genetic and / or epigenetic parameters of the humos gene.
  • the present disclosure further describes a method for producing a diagnostic for the diagnosis of humic-associated diseases by analyzing methylation patterns of the humos gene, wherein the diagnostic agent is characterized in that at least one nucleic acid, optionally together with suitable additives and excipients for its production is used.
  • the present disclosure describes a diagnostic for human-associated diseases by analysis of methylation patterns of the humos gene comprising at least one nucleic acid, optionally together with suitable additives and excipients.
  • the present disclosure further describes the diagnosis and / or prognosis of adverse events for patients or individuals in which the significant genetic and / or epigenetic parameters obtained by the invention within the humos gene can be compared to a different set of genetic and / or epigenetic parameters and the differences thus obtained serve as a basis for diagnosing and / or prognosing adverse events for patients or individuals.
  • hybridization in the context of the present invention is to be understood as meaning a bond forming a duplex structure of an oligonucleotide to a completely complementary sequence in the sense of the Watson-Crick base pairings in the sample DNA.
  • stringent hybridization conditions are meant those conditions in which a hybridization at 60 ° C in 2.5 x SSC buffer followed by several washes at 37 ° C occurs in a lower buffer concentration and remains stable.
  • Genetic parameters in the sense of this invention are mutations and polymorphisms of the humos gene and for its regulation furthermore required sequences.
  • polymorphisms can also be insertions, deletions or inversions.
  • epigenetic parameters are, in particular, cytosine methylations and further chemical modifications of DNA bases of the humos gene and furthermore sequences required for its regulation.
  • Further epigenetic parameters are, for example, the acetylation of histones, which, however, can not be directly analyzed by the method described, but in turn correlated with DNA methylation.
  • MCL mantle cell lymphoma
  • DLBCL diffuse large B-cell lymphoma
  • CLL / SLL chronic lymphocytic leukemia
  • a high probability of methylation corresponds to a low probability of green signals (lighter in the figure) and black mean values of red signals (darker in the figure).
  • the samples on the left side of Figure 1 (A) are assigned to the group of MCL, DLBCL and CLL / SLL, those on the right (B) FL I and II (see also Example No. 3).
  • Seq. ID No.1 shows the sequence of the chemically pretreated genomic DNA of the gene humos
  • Seq. ID No. 2 shows the sequence of a second chemically pretreated genomic DNA of the gene humos
  • Seq. ID No.3 shows the reverse complementary sequence of Seq. ID 1 of the chemically pretreated genomic DNA of the gene humos
  • Seq. ID No.4 shows the reverse complementary sequence of Seq. ID 2 of the chemically pretreated genomic DNA of the gene humos
  • Seq. ID No.5 shows the sequence of an oligonucleotide for the amplification of humos from Example 1
  • Seq. ID No.6 shows the sequence of a second oligonucleotide for the amplification of humos from Example 1
  • Seq. ID No.7 shows the sequence of an oligonucleotide for hybridizing the amplificate of humos from Example 1
  • Seq. ID No.8 shows the sequence of a second oligonucleotide for the hybridization of the amplificate of humos from Example 1
  • Seq. ID No.9 shows the sequence of a third oligonucleotide for the hybridization of the amplificate of humos from Example 1
  • Seq. ID No.10 shows the sequence of a fourth oligonucleotide for hybridizing the amplificate of humos from Example 1
  • Seq. ID No. 11 shows the sequence of an oligonucleotide for hybridizing the amplificate of humos from Example 1
  • Seq. ID No. 12 shows the sequence of a fifth oligonucleotide for hybridizing the amplificate of humos from Example 1
  • Seq. ID No.13 shows the sequence of an oligonucleotide for the hybridization of the amplificate of humos from Example 1
  • Seq. ID No.14 shows the sequence of a sixth oligonucleotide for the hybridization of the amplificate of humos from Example 1
  • Seq. ID No.15 shows the sequence of a seventh oligonucleotide for the hybridization of the amplificate of humos from Example 1
  • Seq. ID No.16 shows the sequence of an eighth oligonucleotide for the hybridization of the amplificate of humos from Example 1
  • Seq. ID No.17 shows the sequence of an eighth oligonucleotide for the hybridization of the amplificate of humos from Example 2
  • Seq. ID No.18 shows the sequence of an eighth oligonucleotide for the hybridization of the amplificate of humos from Example 2
  • Seq. ID No.19 shows the sequence of an eighth oligonucleotide for the hybridization of the amplificate of humos from Example 2
  • Seq. ID No. 20 shows the sequence of an eighth oligonucleotide for the hybridization of the amplificate of humos from Example 2
  • the following example refers to a fragment of the gene humos in which a particular CG position is examined for its methylation status.
  • Example 1 Carrying out the methylation analysis in the humos gene
  • a genomic sequence using bisulfite (bisulfite, disulfite) is treated in such a way that all cytosines not methylated at the 5-position of the base are changed so as to produce a base different in base pairing behavior, whereas those in the 5-position methylated cytosines remain unchanged.
  • bisulfite in the concentration range between 0.1 and 6 M is used for the reaction, an addition takes place at the non-methylated cytosine bases.
  • a denaturing reagent or solvent and a radical scavenger must be present.
  • Subsequent alkaline hydrolysis then leads to conversion of unmethylated cytosine nucleobases to uracil. This converted DNA serves to detect methylated cytosines.
  • the treated DNA sample is diluted with water or an aqueous solution.
  • a desulfonation of the DNA (10-30 min, 90-100 ° C) is carried out at alkaline pH.
  • the DNA sample is amplified in a polymerase chain reaction, preferably with a heat-resistant DNA polymerase.
  • cytosines of the gene humos here from the promoter region or exon 1, examined.
  • the MHH-Call2, MHH-Call4, BV-173, 380, NALM-6 and REH (all human B cell precursor leukemia) cell lines of CD19 + B cells and CCRF-CEM, Jurkat, Molt -17, P12-Ichikawa (all human T cell leukemia), R.PMI-8402 (human T cell acute lymphoblastic leukemia) and Karpas-299 (human T cell lymphoma) are differentiated from CD4 + CD8 T cells.
  • a defined fragment of length 494 bp is amplified using the specific primer oligonucleotides TTTATTGATTGGGAGTAGGT (Seq. ID 5) and CTAATTTTACAAACATCCTA (Seq. ID 6).
  • This amplicon serves as a probe which hybridizes to an oligonucleotide previously bound to a solid phase to form a duplex structure, for example CCTTACTACGTTAAACTC (SEQ ID 7) or CCTTACTACATTAAACTC (SEQ ID 8), with the cytosine to be detected at position 164 of the amplificate.
  • the methylated cytosine is detected with the oligonucleotide (SEQ ID NO: 7) which has guanine at the respective complementary site, whereas the unmethylated state form represented by a thymine is detected with the oligonucleotide (SEQ ID NO: 8) attached to the corresponding nucleotide complementary site of an adenine is detected.
  • the detection of the hybridization product is based on Cy5 fluorescently labeled primer oligonucleotides used for amplification. Only when in the bisulfite-treated DNA at this point a methylated cytosine has been present, there is a hybridization reaction of the amplified DNA with the oligonucleotide. Thus, the methylation status of the respective cytosine to be examined decides on the hybridization product.
  • Example 2 Carrying out the methylation analysis in the humos gene
  • a genomic sequence using bisulfite (bisulfite, disulfite) is treated in such a way that all cytosines not methylated at the 5-position of the base are changed so as to produce a base different in base pairing behavior, whereas those in the 5-position methylated cytosines remain unchanged.
  • bisulfite bisulfite, disulfite
  • an addition takes place at the non-methylated cytosine bases.
  • a denaturing reagent or solvent and a radical scavenger must be present.
  • Subsequent alkaline hydrolysis then leads to conversion of unmethylated cytosine nucleobases to uracil. This converted DNA serves to detect methylated cytosines.
  • the treated DNA sample is diluted with water or an aqueous solution. Preference is then given to desulfonation of the DNA.
  • the DNA sample is amplified in a polymerase chain reaction, preferably with a heat-resistant DNA polymerase. The PCR reactions were carried out in a thermocycler (Eppendorf GmbH). 10 ng of DNA, 0.08 ⁇ M of each primer oligonucleotide, 1.6 mM dNTPs, and one unit of HotstarTaq were used for a 25 ⁇ l preparation. The remaining conditions were chosen according to the manufacturer's instructions.
  • PCR denaturation was first performed at 96 ° C for 15 minutes followed by 46 cycles (60 seconds at 96 ° C, 45 seconds at 55 ° C and 75 seconds at 65 ° C) and a final elongation of 10 minutes at 72 ° C. The presence of the PCR products was checked on agarose gels.
  • cytosines of the gene humos are examined.
  • MCL mantle cell lymphoma
  • DLBCL diffuse large B-cell lymphoma
  • CLL / SLL chronic lymphocytic leukemia
  • a defined fragment of length 523 bp is amplified using the specific primer oligonucleotides TGATTGGGAGTAGGTGTGTT (SEQ ID 17) and CAAATCTTCCAACTTCTCAAA (SEQ ID 18).
  • This amplicon serves as a probe that hybridizes to an oligonucleotide previously bound to a solid phase to form a duplex structure, such as TATGGAGTTCGGTGGTAA (SEQ ID NO: 19) or TATGGAGTTTGGTGGTAA (SEQ ID NO: 20), with the cytosine to be detected at position 259 of the amplicon.
  • the methylated cytosine is mixed with the oligonucleotide ID 19) having a guanine at the respective complementary site, whereas the unmethylated state form represented by a thymine is detected with the oligonucleotide (SEQ ID NO: 20) having an adenine at the respective complementary site , is demonstrated.
  • the detection of the hybridization product is based on CY5 fluorescently labeled primer oligonucleotides used for amplification. Only when in the bisulfite-treated DNA at this point a methylated cytosine has been present, there is a hybridization reaction of the amplified DNA with the oligonucleotide. Thus, the methylation status of the respective cytosine to be examined decides on the hybridization product.
  • MCL mantle cell lymphoma
  • DLBCL diffuse large B-cell lymphoma
  • CLL / SLL chronic lymphocytic leukemia
  • FL I well differentiated follicular lymphoma I
  • FL II moderate differentiated follicular lymphoma II
  • Each of these detection oligonucleotides was designed to hybridize to CpG sites on bisulfite converted sequences that were either unmethylated (TG) or methylated (CG) in their original state.
  • the hybridization conditions were selected to detect differences in single nucleotides of variants TG and CG.
  • the ratios of the two signals were calculated based on the comparison of the intensities of the fluorescent signals.
  • the information is then determined in a weighted matrix (see Figure 1) for CpG methylation differences between two classes of tissues.
  • the most significant CpG positions are displayed at the bottom of the matrix, and the significance decreases at the top.
  • Dark gray in the original figure: red
  • light gray in the original figure: green
  • black a low degree of methylation and black.
  • Each row represents a specific CpG position in a gene and each column shows the methylation profile of different CpGs for a sample.
  • On the left side a gene identification number and a CpG are shown; the associated gene name can be found in Table 1.
  • Table 1 also lists the corresponding accession numbers of the genes.
  • the number before the colon denotes the Gene names and the number behind the colon the specific oligonucleotide.
  • the p-values of the individual CpG positions are shown. The p-values represent the probabilities that the observed distribution happens randomly or not.
  • the first (left in Figure 1) group contains 42 samples of both sexes versus 38 samples of the second (located in Figure 1 on the right) group.
  • the p-value weighted methylation shows a clear distinction between the two groups, 9 CpG positions (red, or gray color shading) of 7 different genes are significantly differentiated (corrected p-value ⁇ 0.05) between the two groups.
  • the cross-validated accuracy of the classification, by SVM (support vector machine) F. Model, P. Adorjan, A. Olek, C. Piepenbrock, Feature selection for DNA methylation based cancer classification., Bioinformatics., 2001 Jun; 17 Suppl 1: S157- 64) is calculated as 78.8% with a species deviation of 2.8%.
  • the humos gene was examined, which is represented by the gene identification number 89.
  • Table 1 Gene number Gene name Accession number 82 EGR4 NM_001965 87 AR NM_000044 88 CDK4 NM_000075 89 HUMOS NM_005372 96 RB1 NM_000321 130 GPIbbeta NM_000407 168 MYOD1 NM_002478 2013 BCL2 NM_000633 2033 CDKN1A NM_000389 2036 CDKN2B NM_004936 2157 MLH1 NM_004936 2267 TGFBR2 NM_003242 2322 TP73 NM_005427 2350 CDKN1C NM_000076 2494 BAK1 NM_001188

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Hospice & Palliative Care (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Oncology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Claims (17)

  1. Procédé pour la détermination de paramètres génétiques et/ou épigénétiques, approprié au diagnostic de maladies existantes ou d'une prédisposition à des maladies définies par analyse de méthylations de cytosine de gêne humain, caractérisé en ce que les étapes suivantes sont exécutées :
    a) dans un échantillon d'ADN génomique, par traitement avec une solution d'un bisulfite, sulfite d'hydrogène ou disulfite et hydrolyse alcaline consécutive en position 5, des bases cytosine non méthylées sont converties en uracile ou en une autre base dont le comportement d'appariement basique est différent de la cytosine ;
    b) des fragments d'ADN prétraité sont amplifiés en recourant à des ensembles d'au moins deux oligonucléotides primaires comprenant chacun au moins une séquence de base longue d'au moins 9 nucléotides, hybridée sur un ADN chimiquement prétraité de gêne humain suivant une des séquences ID NO 1 à ID NO 4, pour l'amplification de séquences ADN d'une des séquences ID NO 1 à ID NO 4 ou de tronçons de celle-ci et à une polymérase,
    c) les amplificats sont hybridés sur un ensemble d'oligomères comprenant au moins une séquence de base longue d'au moins 9 nucléotides, hybridée sur un ADN chimiquement prétraité de gêne humain suivant une des séquences ID NO 1 à ID NO 4, ou sur un groupement de ces oligomères ; et
    d) les amplificats sont ensuite détectés.
  2. Procédé selon la revendication 1, caractérisé en ce que les amplificats portent un marquage détectable.
  3. Procédé selon la revendication 1 ou 2, caractérisé en ce que plus de dix fragments différents sont amplifiés, longs de 100 à 2000 paires de base.
  4. Procédé selon l'une des revendications 1 à 3, caractérisé en ce que l'amplification de plusieurs tronçons d'ADN est exécutée dans un récipient à réaction.
  5. Procédé selon l'une des revendications 1 à 4, caractérisé en ce que la polymérase est une ADN polymérase résistante à la chaleur.
  6. Procédé selon l'une des revendications 1 à 5, caractérisé en ce que les marquages des amplificats sont choisis parmi des marquages fluorescents, des radionucléides ou des fragments moléculaires séparables à masse typique, détectés dans un spectromètre de masse.
  7. Procédé selon l'une des revendications 1 à 6, caractérisé en ce que les amplificats ou des fragments des amplificats sont détectés dans un spectromètre de masse.
  8. Procédé selon l'une des revendications 6 ou 7, caractérisé en ce que pour une détectabilité améliorée dans le spectromètre de masse, les fragments générés présentent une seule charge nette positive ou négative.
  9. Procédé selon l'une des revendications 6 à 8, caractérisé en ce que la détection est exécutée et visualisée par spectrométrie de masse à désorption/ionisation laser assurée par matrice (MALDI) ou par spectrométrie de masse à ionisation électrospray (ESI).
  10. Procédé selon l'une des revendications 1 à 9, caractérisé en ce que l'ADN génomique a été obtenu à partir de cellules ou de composants cellulaires contenant de l'ADN, les sources d'ADN telles que lignes cellulaires, biopsies, sang, expectorations, selles, urine, liquide cérébro-spinal, tissu enrobé de paraffine, p. ex. tissu oculaire, intestinal, rénal, cérébral, cardiaque, prostatique, pulmonaire, mammaire ou hépatique, comprenant des porte-objets histologiques et toutes les combinaisons possibles de ceux-ci.
  11. Procédé selon l'une des revendications 1 à 10, caractérisé en ce qu'il est distingué entre le groupe de lymphome à cellules du manteau (MCL), de lymphome B diffus à grandes cellules (DLBCL), de leucémie lymphatique chronique (CLL/SLL), et le groupe de lymphome I folliculaire (FL I) et de lymphome II folliculaire (FL II).
  12. Procédé selon l'une des revendications 1 à 11, la séquence de base de l'oligomère de l'étape c) comprenant au moins un dinucléotide CpG.
  13. Amplificat, produit par
    a) prétraitement de bases cytosine non méthylées converties en position 5 en uracile ou en une autre base dont le comportement d'appariement basique est différent de la cytosine dans un échantillon d'ADN génomique, par traitement avec une solution d'un bisulfite, sulfite d'hydrogène ou disulfite et hydrolyse alcaline consécutive, et
    b) amplification PCR de fragments d'ADN prétraité en recourant à des ensembles d'oligonucléotides primaires comprenant chacun au moins une séquence de base longue d'au moins 18 nucléotides, inversement complémentaires ou identiques à un ADN chimiquement prétraité de gêne humain suivant une des séquences ID NO 1 à ID NO 4 ou qui sont des tronçons de celle-ci, et à une polymérase, les fragments présentant une longueur de 100 à 2000 paires de base.
  14. Amplificat selon la revendication 13, portant un marquage détectable.
  15. Amplificat selon la revendication 13 ou 14, utilisable pour le diagnostic de maladies.
  16. Utilisation d'un réseau ADN et/ou PNA, lié à une surface de phase solide, d'un ensemble d'oligomères comprenant au moins une séquence de base longue d'au moins 9 nucléotides, identique à un ADN chimiquement prétraité de gêne humain suivant une des séquences ID NO 1 à ID NO 4, ou de fragments de celui-ci pour l'analyse de maladies en relation avec l'état de méthylation du gêne humain.
  17. Utilisation d'un réseau selon la revendication 16 dans un procédé de distinction entre le groupe de lymphome à cellules du manteau (MCL), de lymphome B diffus à grandes cellules (DLBCL), de leucémie lymphatique chronique (CLL/SLL), et le groupe de lymphome I folliculaire (FL I) et de lymphome II folliculaire (FL II).
EP20010982464 2000-11-06 2001-11-06 Diagnostic de maladies associees au gene c-mos humain Expired - Lifetime EP1339873B1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE2000154972 DE10054972A1 (de) 2000-11-06 2000-11-06 Diagnose von mit humos assoziierten Krankheiten
DE10054972 2000-11-06
PCT/EP2001/012831 WO2002036604A2 (fr) 2000-11-06 2001-11-06 Diagnostic de maladies associees au gene c-mos humain

Publications (2)

Publication Number Publication Date
EP1339873A2 EP1339873A2 (fr) 2003-09-03
EP1339873B1 true EP1339873B1 (fr) 2007-01-03

Family

ID=7662311

Family Applications (1)

Application Number Title Priority Date Filing Date
EP20010982464 Expired - Lifetime EP1339873B1 (fr) 2000-11-06 2001-11-06 Diagnostic de maladies associees au gene c-mos humain

Country Status (6)

Country Link
US (1) US20040048278A1 (fr)
EP (1) EP1339873B1 (fr)
AT (1) ATE350489T1 (fr)
AU (1) AU2002214041A1 (fr)
DE (2) DE10054972A1 (fr)
WO (1) WO2002036604A2 (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7617163B2 (en) * 1998-05-01 2009-11-10 Health Discovery Corporation Kernels and kernel methods for spectral data
AU2001278420A1 (en) 2000-04-06 2001-11-07 Epigenomics Ag Diagnosis of diseases associated with dna repair
DE10128508A1 (de) 2001-06-14 2003-02-06 Epigenomics Ag Verfahren und Nukleinsäuren für die Differenzierung von Prostata-Tumoren
US7932027B2 (en) 2005-02-16 2011-04-26 Epigenomics Ag Method for determining the methylation pattern of a polynucleic acid
EP1693468A1 (fr) 2005-02-16 2006-08-23 Epigenomics AG Procédé de détection de l'état de méthylation d'un acide polynucléique
WO2006113770A1 (fr) 2005-04-15 2006-10-26 Epigenomics Ag Procede destine a fournir des fragments d'adn derives d'un echantillon a distance
WO2019068008A1 (fr) 2017-09-30 2019-04-04 Certainteed Gypsum, Inc. Plaques de plâtre effilées et leurs procédés de fabrication

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU766544B2 (en) * 1998-06-06 2003-10-16 Genostic Pharma Limited Probes used for genetic profiling
WO1999064627A2 (fr) * 1998-06-06 1999-12-16 Genostic Pharma Limited Sondes utilisees pour profilage genetique
DE19905082C1 (de) * 1999-01-29 2000-05-18 Epigenomics Gmbh Verfahren zur Identifikation von Cytosin-Methylierungsmustern in genomischen DNA-Proben
DE10128508A1 (de) * 2001-06-14 2003-02-06 Epigenomics Ag Verfahren und Nukleinsäuren für die Differenzierung von Prostata-Tumoren
EP1502222A2 (fr) * 2001-07-02 2005-02-02 Epigenomics AG Systeme distribue pour prevision a base epigenetique de phenotypes complexes

Also Published As

Publication number Publication date
US20040048278A1 (en) 2004-03-11
EP1339873A2 (fr) 2003-09-03
DE10054972A1 (de) 2002-06-06
AU2002214041A1 (en) 2002-05-15
DE50111824D1 (de) 2007-02-15
WO2002036604A2 (fr) 2002-05-10
WO2002036604A3 (fr) 2002-08-08
ATE350489T1 (de) 2007-01-15

Similar Documents

Publication Publication Date Title
EP1370691B1 (fr) Procede de detection de structures de methylation de la cytosine a haute sensibilite
DE10130800B4 (de) Verfahren zum Nachweis von Cytosin-Methylierung mit hoher Sensitivität
DE60126593T2 (de) Diagnose von mit apoptose assoziierten erkrankungen mittels ermittlung des methylierungszustandes von apoptose-assozierten genen
EP1423528B1 (fr) Procede de determination du degre de methylation de cytosines determinees d'adn genomique dans le contexte sequentiel 5'-cpg-3'
DE10151055B4 (de) Verfahren zum Nachweis von Cytosin-Methylierung in CpG Inseln
EP1463841A2 (fr) Procede de detection de modeles de methylation de la cytosine par ligation exponentielle d'oligonucleotides sondes hybrides (mla)
EP1438437B1 (fr) Procede de detection de methylation de la cytosine dans des echantillons d'adn immobilise
DE10132212B4 (de) Verfahren zum Nachweis von Cytosin-Methylierung durch vergleichende Analyse der Einzelstränge von Amplifikaten
DE10161625A1 (de) Verfahren und Nukleinsäuren für die Analyse einer Lungenzell-Zellteilungsstörung
DE10128508A1 (de) Verfahren und Nukleinsäuren für die Differenzierung von Prostata-Tumoren
DE20121960U1 (de) Nukleinsäuren für die Diagnose von mit Metastase assoziierten Krankheiten
EP1339873B1 (fr) Diagnostic de maladies associees au gene c-mos humain
WO2002036814A2 (fr) Diagnostic de maladies associees au gene cdk4
DE10304219B3 (de) Verfahren zum Nachweis von Cytosin-Methylierungsmustern mit hoher Sensitivität
EP1432827B1 (fr) Procede pour detecter la methylation de l'adn au moyen d'analogues marques de la s-adenosylmethionine
DE20121967U1 (de) Nukleinsäuren für die Diagnose von Verhaltensstörungen, neurologischen Erkrankungen und Krebs
DE20121965U1 (de) Nukleinsäuren für die Diagnose von mit DNA Addukten assoziierten Krankheiten
DE10230692A1 (de) Verfahren und Nukleinsäuren für die Analyse von Methylierungsmustern innerhalb des DD3-Gens
DE10255104A1 (de) Verfahren und Nukleinsäuren für die Analyse von proliferativen Erkrankungen von Brustzellen
DE20121964U1 (de) Nukleinsäuren für die Diagnose von mit dem Metabolismus assoziierten Krankheiten
DE10164501A1 (de) Verfahren und Nukleinsäuren für die Analyse einer Lymphoid-Zellteilungsstörung
DE20121961U1 (de) Nukleinsäuren für die Diagnose von mit der Signaltransduktion assoziierten Krankheiten
DE20121977U1 (de) Nukleinsäuren für die Diagnose von mit Tumor-Suppressorgenen und Onkogenen assoziierten Krankheiten
DE20121968U1 (de) Nukleinsäuren für die Diagnose von mit Apoptose assoziierten Krankheiten
DE20121974U1 (de) Nukleinsäuren für die Diagnose von mit DNA Replikation assoziierten Krankheiten

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20030506

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

17Q First examination report despatched

Effective date: 20040213

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: EPIGENOMICS AG

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070103

Ref country code: IE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070103

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070103

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070103

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

Free format text: NOT ENGLISH

REF Corresponds to:

Ref document number: 50111824

Country of ref document: DE

Date of ref document: 20070215

Kind code of ref document: P

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

Free format text: LANGUAGE OF EP DOCUMENT: GERMAN

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070403

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070414

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070604

NLV1 Nl: lapsed or annulled due to failure to fulfill the requirements of art. 29p and 29m of the patents act
GBV Gb: ep patent (uk) treated as always having been void in accordance with gb section 77(7)/1977 [no translation filed]

Effective date: 20070103

REG Reference to a national code

Ref country code: IE

Ref legal event code: FD4D

EN Fr: translation not filed
PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070103

26N No opposition filed

Effective date: 20071005

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070103

Ref country code: FR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070824

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070404

BERE Be: lapsed

Owner name: EPIGENOMICS A.G.

Effective date: 20071130

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20071130

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20071130

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20071130

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20071130

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20080603

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070103

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20071106

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070103

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20071106

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070103